Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been successfully used in measuring intact protein molecular masses, identifying post-translational modifications of proteins, elucidating structure domains of proteins, and identifying unknown proteins. In most of these applications, mass (exactly should be mass/charge) is the only information being utilized. The peak intensity as the second part information provided by mass spectrometry is hardly used because the complexity of matrix-assisted laser desorption ionization processes. From a practical point view, we investigated the effects of matrix, solvent on peak intensity as well as sample distribution on sample probe of proteins and peptides with different size and different hydrophobicity. Based on these investigations, we formalized an experimental protocol on how to select solvent, select internal standard, and collect spectra for quantitative analysis of prote in and pep tide using MALDI-TOF-MS. The plot of relative peak intensity vs. protein concentration showed linear range of three to four orders of magnitude for quantitation. Our data indicated that the coefficient variation of quantitation by MALDI can be limited to less than 20%. The quantitative analysis of protein by MALDI-TOF-MS has its own advantages comparing with other mass spectrometric method, which all of the ions of protein and peptide are detected in one spectrum and the concentration of these protein and peptide can be measured at once.